Compositions and Methods for Generation of Infectious Hepatitis C Virus in Immortalized Human Hepatocytes

ABSTRACT

The present invention provides a cell line capable producing infectious hepatitis C virus 1a (HCV 1a) particles in culture. Disclosed are compositions and methods for an HCV 1a (clone H77) transfected immortal human hepatocyte (IHH) capable of generating infectious HCV 1a virus particles in culture. Also disclosed are methods of using the cell line, or HCV 1a virus particles derived from said cell line, to screen for potential therapeutic agents which interfere with HCV 1a virus propagation to treat hepatic disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of and claims priority toU.S. patent application Ser. No. 11/506,161 filed Aug. 17, 2006,pending. All documents above are incorporated herein in their entiretyby reference.

GOVERNMENT SUPPORT CLAUSE

The work disclosed herein was supported by research grants A145144(R.B.R) and CA85486 (R,R) from the National Institutes of Health. TheU.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to compositions and methods ofpropagating hepatitis C viral genomes and infectious particles.Specifically, the invention is directed to an immortalized human hepaticcell line comprising the hepatitis C viral 1a genome, (IHH/HCV 1a) whichis capable of replicating the hepatitis C viral 1a (HCV 1a) genotype andproducing fully infectious HCV 1a virus particles.

2. Description of the Related Art

According to the American Liver Foundation, over 300,000 Americans arehospitalized each year for cirrhosis of the liver. The primary causes ofcirrhosis are alcohol abuse and chronic hepatitis C(HCV). To date,approximately 3.9 million Americans suffer from hepatitis C. The mostimportant feature of HCV infection is the development of chronichepatitis in a significant number of infected individuals and thepotential for disease progression to cirrhosis and hepatocellularcarcinoma (6, 7, 11, 27). At present, the only approved therapy forchronic HCV infection is interferon (IFN)-α with or without ribavirin(9, 21), but this therapy fails to clear HCV from a significant numberof patients (22). A number of HCV genomes have been cloned, and sequencedivergence indicates several genotypes as well as a series of subtypesfor this virus (28). In the United States, HCV genotypes 1a and 1b arepredominant in patients with chronic hepatitis C (31). Progress in theunderstanding HCV biology has been hampered due to the lack of anefficient cell culture system for virus growth. The establishment ofself-replicating HCV full-length genomic replicons from genotypes 1a and1b in human hepatoma (Huh-7) cells has provided an important tool forthe study of HCV replication mechanisms (3, 10, 23). Although somegroups have reported the generation of infectious virus fromtransfection of genomic RNA of HCV genotype 2a into Huh-7 (5, 15, 29,32), the generation of infectious HCV genotype 1a has not beensuccessful to date, and therefore a long felt need exists.

The inventors and others have previously shown that HCV core proteintranscriptionally regulates a number of cellular genes (26). Theinventors have also previously described the generation of immortalizedhuman hepatocytes (IHH) by transfection of the HCV core genomic regionfrom genotype 1a (2, 25). IHH exhibit a weak level of HCV core proteinexpression, albumin secretion, glucose phosphatase activity, and absenceof smooth muscle actin. IHH also displayed focal cytoplasmic andmembrane staining for carcinoembryonic antigen (CEA), biliaryglycoprotein (BGP1/CEACAM1) and nonspecific cross-reacting antigen(NCA/CEACAM6), and expression of hepato-biliary transport marker genes(MRP, LST1 and NTCP). Together, these results suggested that IHH arewell differentiated. HCV core protein selectively degrades STAT1,reduces phosphorylated STAT1 (P-STAT1) accumulation in the nucleus in aproteasome-dependent manner, and impairs IFN-α-induced signaltransduction via suppressor of cytokine signaling-3 expression (1, 4,16). HCV core protein is competent to partially rescue growth of agenetically engineered influenza A virus lacking its own IFN antagonist(4). The core protein can modulate interferon regulatory factor (IRF),Jak-STAT and inducible nitric oxide synthetase (iNOS) pathways, andsuggest mechanisms by which core could affect HCV persistence andpathogenesis (20). Since HCV core protein transcriptionally regulatesseveral cellular genes involved in cell growth, apoptosis and defensemechanism, the inventors hypothesize that IHH may set the stage for HCVgenome replication and assembly.

The inventors have sought to address the long felt need of providing acell line permissible for HCV 1a replication and generation ofinfections virus particles. A cell line with this capability will beinvaluable to researchers not only by providing easy access to HCV 1ainfectious virus particles, but to identify HCV mediators and theirpathways. In addition, it will be invaluable as a tool to screen newtherapeutic strategies or potential pharmacological agents whichinterfere with the propagation of HCV and resulting diseases caused bythe virus.

SUMMARY OF THE INVENTION

Progress in the understanding hepatitis C virus (HCV) biology hasremained challenging due to the lack of an efficient cell culture systemfor virus growth. Therefore, the inventors examined HCV core proteinmediated immortalized human hepatocytes (IHH) for support of HCV 1agenomic replication, infectious particle generation, and propagation. Invitro transcribed HCV full-length RNA from genotype 1a (clone H77) waselectroporated into IHH. Viral RNA replication was evident by reversetranscriptase-polymerase chain reaction (RT-PCR) analysis of cellularRNA isolated from the HCV 1a genome-transfected IHH (IHH/HCV 1a). HCVfull-length genome transfected IHH also displayed viral proteinexpression as indicated by indirect immunofluorescence. In contrast,cells transfected with polymerase-defective HCV (H77/GND) RNA as anegative control did not exhibit expression of viral genome. Immunogoldlabeling and transmission electronic microscopy demonstratedlocalization of the viral E1 protein in the rough endoplasmic reticulumof RNA transfected IHH. Virus-like particles of ˜50 nm in the cytoplasmwere also observed. Naive IHH, inoculated with culture medium from HCVfull-length genome transfected cells, displayed NS5a protein expressionin a dilution dependent manner, which was reduced upon prior incubationof the inoculums with HCV infected patient serum. Based on NS5a positiveimmunofluorescence, the inventors determined ˜4.5×10⁴-1×10⁵ fluorescentfocus units (ffu)/ml of cell culture medium, removed from cultures ofIHH transfected with H77 full-length RNA. A similar level of virusgrowth was observed by transfection of RNA from HCV genotype 2a (JFH1)into IHH. Taken together, our results suggest that IHH support HCVgenome replication and virus assembly.

Therefore, an object of this invention is an immortal human hepatocytecomprising a full length HCV 1a genome (IHH/HCV 1a) capable ofsustaining HCV 1a replication and generation of infectious HCV 1a virusparticles.

In another embodiment, an object of this invention is an infectious HCV1a virus particle produced by IHH/HCV 1a in culture.

In another embodiment, an object of this invention is a method of usingIHH/HCV 1a to study HCV 1a biology and to screen for potentialtherapeutic agents which interfere with any stage of HCV propagation.

In another embodiment, IHH/HCV 1a derived HCV 1a virus particles may beused to study infectivity and screen therapeutic agents which mayinterfere with HCV 1a infection.

It is envisioned that the instant IHH/HCV 1a will be propagated invitro, frozen for convenient storage, and typically manipulated as aconventional cell line.

It is also envisioned that IHH/HCV 1a, its prodigy, or derivatives, inwhole or in part, maybe be used separately, or combined with otherbiological systems to screen for therapeutic agents which interfere withpathways of viral replication, assembly, and infection, for developmentof pharmaceutical compositions for the treatment of hepatic disease.

DESCRIPTION OF THE DRAWINGS

FIG. 1: HCV RNA and protein expression in IHH. Panel A: RT-PCR analysiswas performed using 5′ untranslated region (5′ UTR) specific primersfrom RNA isolated at day 5 from two different sets of IHH transfectedwith H77/GND RNA as a negative control (lanes 1 and 2) and H77 RNA(lanes 3 and 4). GPDH was amplified as an internal control. The sizes ofthe amplified bands were verified from the migration of a φX174-HaeIIIdigested DNA marker (not shown). Panel B: Western blot analysis for coreprotein expression in H77/GND RNA transfected (lane 1), and H77 RNAtransfected (lane 2) IHH, using a specific antiserum. The blot wasre-probed with antibody to actin for similar protein load in each lane.Panel C: Western blot analysis for NS3 protein expression in twodifferent sets of IHH transfected with H77/GND RNA (lanes 1 and 2) andH77 RNA (lanes 3 and 4), using a specific monoclonal antibody. The blotwas re-probed with antibody to actin for similar protein load in eachlane. The molecular weight of the protein bands were verified from themigration of protein molecular weight markers (Cambrex, Rockland, Me.).

FIG. 2: Intracellular expression of HCV proteins. IHH transfected withRNA from H77 clone (panel A) or H77/GND RNA transfected negative control(panel B) were treated with NS5a specific monoclonal antibody fordetection of protein expression by intracellular immunofluorescenceafter 5 days of transfection. IHH were similarly transfected with RNAfrom JFH1 clone (panel C) and JFH1/GND RNA transfected negative control(panel D) for intracellular localization of NS3 using a specificmonoclonal antibody. Green color indicates NS5a staining and red colorfor NS3 staining.

FIG. 3: Immunogold localization of HCV E1 protein and virus-likeparticles in IHH with a specific monoclonal antibody. Panel A:Localization of virus-like particles in the cytoplasm are indicated byarrows. Panel B: Localization of HCV E1 protein to the rough endoplasmicreticulum is marked by arrows. Panel C: Localization of virus-likeparticles in the cortical cytoplasm adjacent to the plasma membrane.Panel D: Localization of virus-like particles in a large phagic vacuolein the cytoplasm of IHH is shown by arrows. Panel E: Clusters of CGindicated by arrows show virus-like particles in IHH. The labeledparticle indicated by an arrow and with an asterisk is shown at highermagnification in the inset. As observed by light microscopy, IHH containcytoplasmic vacuoles and lipid droplets. Panel F: H77/GNDRNA-transfected control section of IHH incubated with monoclonalantibody to E1 glycoprotein did not exhibit immunogold labeling. Othernegative controls consisted of labeling with normal mouse IgG andomitting the primary antibody (not shown). The abbreviations used are:C—cytoplasm, M—mitochondrion, PM—plasma membrane, RER—rough endoplasmicreticulum, V—vacuole, LD—lipid droplet, PV—phagic vacuole. Magnificationbars are 0.25 μm in panels A-F, and 0.1 μm in the inset in panel E.

FIG. 4: Presence of HCV in culture medium and infectivity of naive IHH.Panel A: RT-PCR analysis was performed for detection of 5′ UTR fromculture medium of HCV RNA transfected IHH. Filtered culture medium fromIHH transfected with H77/GND RNA (lane 1), full-length H77 RNA (lane 2),JFH1/GND (lane 3), full-length JFH1 RNA (lane 4) were analyzed foramplification of 5′ UTR. HCV genome was amplified similarly from Huh-7cells transfected with JFH1/GND RNA (lane 5) and full-length JFH1 RNA(lane 6). Cloned H77 DNA was included as a positive control in PCRamplification (lane 7). Panel B: Immunofluorescence of IHH at day 3after infection with filtered culture medium from H77 (panel a) and JFH1(panel b) for detection of NS5a and NS3 protein expression,respectively. Panel C: Generation of infectious HCV after transfectionof H77 genomic RNA into IHH. In vitro transcribed H77 RNA (2 ug) waselectroporated into 1×10⁶ IHH. HCV RNA copies at the intracellular (Δ-Δ)level and in the culture supernatant (numbers on top) were measured byreal-time PCR at indicated days. Virus infectivity of the culturesupernatant was determined in naive IHH and is expressed as ffu/ml(black bars). Panel D: Neutralization of virus infectivity by HCVinfected patient serum (black bars). Two fold serial dilutions of testserum was incubated with ˜100 fluorescent focus unit of virus generatedfrom H77 clone at 37° C. for 30 minutes. Virus-serum mixture was addedon naive IHH and incubated for 3 days for determination of fluorescentfocus unit by indirect immunofluorescence using NS5a specific antibody.Similar experiment was performed in parallel with serum from a healthyindividual (hatched bars). The results are presented as % inhibition offluorescent focus unit, and variation from triplicate assays areindicated by error bars.

DETAILED DESCRIPTION OF THE INVENTION

HCV replication is believed to proceed as follows. HCV RNA is translateddirectly to a precursor viral polypeptide. This precursor polypeptide isthen proteolytically cleaved to form individual proteins. A replicasecomplex amplifies viral RNA genome via a minus strand intermediate. Plusstrand RNA progeny are then packaged into virus particles which acquiretheir envelope by budding into the lumen of the endoplasmic reticulum.HCV particles are exported via the constitutive secretory pathway. Basedon this working principle, the inventors have shown that IHH support HCVgenome replication and protein expression from genotype 1a.

Transmission electron microscopy and immunogold labeling, using amonoclonal antibody directed against HCV E1 glycoprotein, demonstratedthe localization of this antigen to the rough endoplasmic reticulum, andalso the formation of virus-like particles. The inventors transferredculture medium, previously in contact with IHH/HCV 1a, to naive IHHcultures and subsequently detected HCV infection in these cells byRT-PCR and indirect immunofluorescence.

The inventors observed JFH1 replication and virus assembly in IHH. Theinfectious unit of JFH1 replicated in IHH similarly to JFH1 grown inHuh-7 cells or its derivatives. The inventors also observed similarlevels of genomic copy of H77 or JFH1, in transfected IHH culturesupernatant, and in fluorescence focus units. The inventors did notpurify virus particles for negative staining due to the relatively lowinfectious units present in the culture medium.

Three different groups of investigators have reported differentdensities of HCV 2a particles. Zhong et al. (32) observed peakinfectivity from an apparent density of 1.105 gm/ml, Wakita et al. (29)observed peak infectivity at a density of 1.15 gm/ml, and Lidenbach etal. (15) observed a broad distribution of virus infectivity over a rangeof 1.01 to 1.12 gm/ml. A similar finding suggesting a variation ofbuoyant density of cell culture grown HCV 2a between 1.06 and 1.16 gm/mlwas reported by Cai et al. (5). HCV is known to associate with serumimmunoglobulin and lipoproteins (24). The inventors found HCVinfectivity within a density range of 1.09 to 1.12 sucrose gradient,which did not correlate with the highest copy number of virus genomicRNA (data not shown).

Recently, HCV production was reported from a HCV-ribozyme construct ofgenotype 1a (clone H77) in Huh-7 cells, although infectivity of viruswas not demonstrated (8). Virus genome replication and assembly aremulti-step processes, and are influenced by the intracellular milieu.Inhibition of host cell growth and induction of cytokines, such asinterferons, may have an impact on prevention of virus replication (3).The inventors provide evidence for HCV replication and assembly ofinfectious genotype 1a in IHH. Others have not been successful ingenerating infectious HCV 1a from cells in culture.

The inventors speculate that the cellular defense mechanism against HCVinfection is attenuated or compromised in IHH. The inventors realize theimportance of determining these factors including mechanisms for growthof HCV in IHH, as well as identification of critical control points inthe HCV life-cycle. The inventors currently have studies in progress todetermine cellular and viral factors influencing virus growth, such asserial passage requirements for adaptation in IHH, mutations at specificsites of the HCV genome, and selection of cell populations forattenuation protective mechanisms. The inventors realize the importanceof further characterizing biophysical properties of cell culture grownHCV 1a, including infectivity in appropriate animal models.

Therefore, the invention is drawn to (1) an immortal human hepatocytecell line comprising a full length HCV 1a genome, capable of producinginfectious HCV 1a virus particles, (2) methods of producing said cellline (3) methods of producing infectious HCV 1a virus particles, (4)methods of using said cell line for evaluating potential therapeuticagents for the prevention of HCV 1a propagation, (5) methods of usingcell culture derived HCV 1a for evaluating potential therapeutic agentsfor the prevention infection.

The term “HCV” refers to hepatitis type C virus.

The term “1a”, or “HCV 1a” refers to the 1a genotype of the hepatitis Cvirus.

The term “immortalized human hepatocytes” or “IHH” means generally acell line derived from human hepatocytes and modified to be maintainedindefinitely in vitro. More specifically it refers to an IHH, made bythe inventors and previously fully described by them (2, 25) which areherein incorporated by reference.

The term “H77” refers to full length HCV 1a genomic oligonucleotide, asprovided by the H77 clone previously described in detail (3, 13, 14)which are herein incorporated by reference.

The term IHH/HCV 1a refers to the instance invention of an IHH cellcomprising a full lengthen HCV 1a genome capable of producing HCV 1ainfectious virus particles.

The term “propagation” in reference to HCV refers to the process inwhole or in part of infection or transfection, oligonucleotidereplication, protein synthesis, virus particle assemble, release andre-infection.

Sequence identity or percent identity is intended to mean the percentageof same residues between two sequences. The reference sequence isHepatitis C Viral Genome 1a accession number NC_(—)004102 which isderived from AF009606. In all of the sequence comparisons, the twosequences being compared are aligned using the Clustal method (Higginset al, Cabios 8:189-191, 1992) of multiple sequence alignment in theLasergene biocomputing software (DNASTAR, INC, Madison, Wis.). In thismethod, multiple alignments are carried out in a progressive manner, inwhich larger and larger alignment groups are assembled using similarityscores calculated from a series of pairwise alignments. Optimal sequencealignments are obtained by finding the maximum alignment score, which isthe average of all scores between the separate residues in thealignment, determined from a residue weight table representing theprobability of a given amino acid change occurring in two relatedproteins over a given evolutionary interval. Penalties for opening andlengthening gaps in the alignment contribute to the score. The defaultparameters used with this program are as follows: gap penalty formultiple alignment=10; gap length penalty for multiple alignment=10;k-tuple value in pairwise alignment=1; gap penalty in pairwisealignment=3; window value in pairwise alignment=5; diagonals saved inpairwise alignment=5. The residue weight table used for the alignmentprogram is PAM250 (Dayhoff et al., in Atlas of Protein Sequence andStructure, Dayhoff, Ed., NBRF, Washington, Vol. 5, suppl. 3, p. 345,1978).

Table 1 shows the calculations of identity for comparisons of HCVderived from H77.

TABLE 1 Percent Identity of HCV H77 derived sequences Species Accessionnumber Percent Identity Hepatitis C Viral NC_004102 100 Genome 1a (H77)Hepatitis C virus AF009606 100 polyprotein gene, complete cds 1aHepatitis C virus strain AF011752 99 H77 pCV-H11

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims, which follow the examples.

EXAMPLE 1 Replication of HCV Genome and Virus Protein Expression

The object of the invention is a cell line to provide HCV 1a genotypereplication and generation of infectious virus particles. The inventorshave previously described the generation of immortalized humanhepatocytes (IHH) by transfection of HCV core genomic region fromgenotype 1a, as well as the conditions and requirements for maintainingIHH in culture (2, 25) which are herein incorporated by reference.Full-length RNAs from HCV genotype 1a (clone H77, GenBank Accessionnumber NC_(—)004102, SEQ ID NO:1) has also previously described (3, 13,14) and are herein incorporated by reference. Restriction enzyme Xba Iand T7 RNA polymerase were obtained from Invitrogen and Promega,(Madison, Wis.) respectively and used according to the supplier'sprotocol. The individual elements of the inventors' methodology isgenerally well known and described in detail in numerous laboratoryprotocols, one of which is Molecular Cloning 2^(nd) edition, (1989)Sambrook, J., Fritsch, E. F. and Maniatis, J., Cold Spring Harbor.

Clone H77 contains a 5′ untranslated region (5′ UTR) coding sequence and3′ UTR, which is suggested to be necessary for replication (14, 30). Invitro transcribed full-length HCV 1a RNA from clone H77 was used fortransfection of IHH which was performed by electroporation. Polymerasedefective H77/GND RNA was similarly used as a negative control. H77 cDNAwas first linearized by digestion with Xba I, and the linearized productpurified by agarose gel electrophoresis. Purified H77 cDNA was thentranscribed in vitro using T7 RNA polymerase. In vitro transcribed RNA(1-2 μg) was introduced into 5×10⁶ IHH by electroporation (950 μF and270 V) using a BioRad Gene pulse Xcell system (Hercules, Calif.). Thetransfected cells were plated on collagen coated plastic dishes, andmaintained using standard cell culture techniques to allow for HCVreplication.

Total cellular RNA was extracted 5 days post transfection. For detectionof HCV genome, total cellular RNA and random hexamer were used for cDNAsynthesis with a SuperScript III first-strand synthesis system(Invitrogen), according to the supplier's protocol. PCR amplificationwas performed with cDNA as a template, using sense(5′-ACCCGCTGAATTCCTGGAGA-3′) (SEQ ID NO:2) and antisense(5′-CACGGTCTTCTAGACCTCCC-3′) (SEQ ID NO:3) primers from 5′ UTR, at 94°C. for 30s, annealing at 55° C. for 60s, and extension at 72° C. for90s. GPDH was used as an internal control, with specific primers (17).RT-PCR analyses suggested amplification of 120 bp sequence from the 5′UTR (FIG. 1, panel A). In contrast, cells transfected with H77/GND RNAdid not exhibit the presence of HCV genomic sequence. To rule out theintegration of H77 plasmid DNA into IHH, the genomic DNA from cell lineswere isolated and examined for HCV genome by PCR. These resultssuggested the absence of HCV sequence, indicating HCV genomic RNAreplication in the cytoplasm of IHH (data not shown). Filtered culturesupernatant was also treated with RNaseA prior to isolation of viralRNA. RT-PCR was performed for NS5A region (17) and the inventors haveobserved amplification of specific RNA sequence.

Western blot analysis was performed to analyze the expression of coreand NS3 proteins in control and experimental cells using specificantibodies. An equal amount of proteins from whole cell lysates insample buffer were separated by SDS-PAGE. Proteins were transferred ontonitrocellulose, incubated with specific antibodies, and detected bychemiluminescence (Amersham, Piscataway, N.J.). HCV core protein wasdetected by a specific rabbit antiserum, and NS3 was detected by aspecific mouse monoclonal antibody (Virogen, Watertown, Mass.). Blotswere stripped and re-probed using a mouse monoclonal antibody to actin(Oncogene Science, Cambridge, Mass.). IHH supporting HCV genomereplication displayed the presence of core (˜21 kDa) and NS3 (˜63 kDa)proteins (FIG. 1, panels B and C). On the other hand, IHH transfectedwith H77/GND RNA did not show a detectable level of core or NS3proteins. A weak level of core protein was detected in this set of IHHfor immortalization by HCV core protein (FIG. 1, panel B). IHHtransfected with HCV full-length RNA were passaged at 4 or 5 dayintervals. HCV RNA and protein expression were detected up to 12 days ofcell culture, and discontinued for the lack of growth after 2 weeks.

To further examine intracellular expression of HCV protein, IHHtransfected with H77 RNA were fixed with 3.7% formaldehyde and incubatedat room temperature for 1 h with monoclonal antibodies to NS5a(Biogenesis, Kingstone, N.H.). Cells were washed three times with PBSand stained with anti-mouse Ig conjugated with Alexa 568 (MolecularProbes, Eugene, Oreg.), and mounted for fluorescence microscopy. Primaryantibodies and secondary antibody-fluorochrome conjugates were titratedfor use of optimum dilutions where there was no background fluorescence.The inventors have observed cytoplasmic expression of NS5a (FIG. 2,panel A) in 60% IHH after 5 days of transfection. HCV genotype 2a (cloneJFH1) has been shown to grow in Huh-7 cells or its derivatives (5, 15,29, 32). In vitro transcribed RNA from clone JFH1 was used fortransfection of IHH to determine if the immortalized hepatocyte cellline supports HCV growth. Intracellular localization of NS3 protein fromJFH1 RNA transfected IHH was detected by immunofluorescence (FIG. 2,panel C). The inventors have also used Huh-7.5 cells transfected withJFH1 RNA as a positive control (29) and observed NS3 expression byindirect immunofluorescence (data not shown). On the other hand, IHHsimilarly transfected with RNA from H77/GND or JFH1/GND clone did notdisplay virus protein expression by immunofluorescence (FIG. 2, panels Band D).

EXAMPLE 2 Immunogold Localization of Virus-Like Particles

Phase contrast microscopy suggested that HCV genome transfected IHH wereswollen with large vacuoles in the cytoplasm, whereas negative controlsdid not show any detectable changes. The inventors also examinedcellular changes using an electron microscopy, and found at theultrastructural level that some of these vacuoles appeared to be empty(FIG. 3, panels A and E). Others vacuoles contained lipid (FIG. 3, panelE) or material isolated for degradation (FIG. 3, panel D).Ultrastructural changes also included an increased polymorphism ofnuclei (FIG. 3, panel E). Immunogold labeling was performed forlocalization of HCV like particles in transfected IHH. For this,transfected IHH (4 days in culture) were detached from collagen coatedpetridishes by a brief trypsin treatment, pelleted in a microcentrifuge,and fixed in 4% paraformaldehyde and 1% glutaraldehyde in PBS for 16 hat 4° C. After washing with PBS, cells were further washed in distilledwater, dehydrated in ethanol, and infiltrated with L.R. White resin(London Resin Company, Berkshire, UK). The cell pellets were polymerizedin BEEM capsules (Ted Pella, Inc., Redding, Calif.) at −20° C. underultraviolet light. Thin sections were cut from blocks, collected onformvar-coated nickel grids, and blocked with 1% fish gelatin and 1% BSAin PBS for 10 min. Sections were incubated for 2 h in 1:100 dilution(titrated before hand for best results) of monoclonal antibody to E1glycoprotein (305/C3) or normal mouse IgG in PBS containing 0.1% BSA,washed in PBS containing 0.1% BSA and incubated for 1 h in Protein A-10nm colloidal gold (CG) diluted 1:200 in PBS containing 0.1% BSA. Afterwashing with PBS, the grids were fixed for 3 min in glutaraldehyde,washed in distilled water, stained with uranyl acetate and lead citrate,and photographed with a JEOL 100 CX electron microscope. No clusters ofCG particles were observed in controls which consisted of staining withnormal mouse IgG, omitting the primary antibody, or stainingmock-transfected IHH with the 305/C3 monoclonal antibody. Severalhundred cells were evaluated in each case. Immunogold labeling with E1specific monoclonal antibody demonstrated the presence of HCV-likeparticles and E1 protein in IHH. Numerous labeled virus-like particleswere observed in the cytoplasm (FIG. 3, panels A and E) and near theplasma membrane (FIG. 3, panel C) of H77 RNA transfected IHH. Thelabeled particles were 50 nm in diameter. Extensive labeling was alsoassociated with the rough endoplasmic reticulum consistent with thesynthesis of E1 viral protein (FIG. 3, panel B). In addition, theinventors observed cytoplasmic phagic vacuoles which containedgold-labeled virus-like particles (FIG. 3, panel D).

Processing of cells into LR White resin for immunogold localizationomits the conventional osmium tetroxide fixation step to preserveantigenicity but comes at the cost of reduced tissue contrast. Inaddition, the identification of virus particles by immunogold labelingat the ultrastructural level can be problematic. For this, the inventorscarried out a series of control experiments to insure labelingspecificity. First, clusters of CG on virus-like particles, and singleCG particles were observed in the endoplasmic reticulum in severalindependent anti-E1 labeling experiments. Second, H77/GND RNAtransfected IHH (negative controls) showed no such clusters of CG in thecytoplasm or single CG particles localized along the endoplasmicreticulum or membranes (FIG. 3, panel F). Third, sections of HCV genometransfected IHH incubated with normal mouse IgG at similar IgGconcentrations as used for the anti-E1 antibody did not result in anyspecific immunogold labeling. Fourth, omitting anti-E1 antibody did notresult in any specific immunogold labeling. Finally, CG particles in theanti-E1 labeling experiments were primarily confined to cells and werenot observed to any degree in the spaces around cells, again suggestingthe labeling was specific for E1 protein in cells. Thus, the appearanceof virus-like particles in RNA transfected IHH indicates that IHHsupport HCV 1a viral replication and assemble.

EXAMPLE 3 Infection of IHH by HCV from Culture Medium

Inventors next examined the presence of HCV in cell culture medium fromIHH. After different transfection time periods, culture medium wasfiltered through a 0.45 Mm cellulose acetate membrane (Millipore,Bedford, Mass.), and concentrated to 10-20 fold by Milliporeultrafiltration (100 kDa cut off) and used for detection of HCV genomicsequence by RT-PCR (FIG. 4, panel A). The presence of HCV 5′ UTR wasdetected in culture medium from HCV genome transfected IHH, but not frompolymerase defective HCV RNA transfected IHH. The inventors obtained˜1.1×10⁸ genome copies/ml of culture medium using real-time RT-PCR, asrecently described (32). Culture supernatant collected for 7 dayssuggested a peak of HCV genome copy number between 4 and 5 days aftertransfection.

The inventors then determined whether the culture medium containedinfectious HCV. For this, culture medium was serially diluted two foldand inoculated into naive IHH. Cells were incubated for 4 h, washed andincubated with fresh medium for 3 days before analysis of ffu/ml byindirect immunofluorescence for NS5a (H77 clone) or NS3 (JFH1 clone) asrecently described (32). Nuclear staining was performed usingTO-PRO3-iodide (Molecular Probes) and cells were mounted for confocallaser scanning microscopy (Bio-Rad, Model 1024). A representative figuredisplaying infection of IHH by H77 or JFH1 is shown (FIG. 4, panel B).The number of fluorescing cells was counted and correlated withdilutions of cell culture medium for determination of ffu/ml. Theinventors observed a 4.5×10⁴-1×10⁵ ffu/ml of the cell culture mediumafter 5 days of transfection from both H77 and JFH1 clones.

The inventors transfected in vitro transcribed H77 or JFH1 RNA into IHHand isolated RNA from transfected cells. Culture supernatant was alsocollected for isolation of RNA and determination of infectivity(ffu/ml). Real-Time PCR suggested maximal HCV RNA accumulation from H77at the intracellular level on day 2, which declined on day 5 (FIG. 4,panel C). The inventors have observed higher genome copy number andinfectious virus titer at day 4. Similarly, JFH1 RNA transfected IHHsupernatant displayed a peak genome copy number of 10⁸/ml, andinfectivity of ˜7×10⁴ ffu/ml on day 4.

A HCV infected patient serum (OP1843) displaying neutralizing activityagainst VSV/HCV pseudotype (19) was used in determining neutralizationof cell culture grown HCV. Serum from a healthy volunteer was used as anegative control in HCV neutralization assay. A two fold serial dilutionof heat inactivated serum was incubated with ˜100 fluorescent focusunits of HCV generated from H77 clone at 37° C. for 30 minutes.Virus-serum mixture was added to naive IHH cultures and incubated for 3days. Neutralization of fluorescent focus unit was determined from theinhibition of NS5a protein expression by immunofluorescence. Results areshown as percent inhibition of fluorescent focus unit (FIG. 4, panel D).A 60% infectivity was inhibited upon prior incubation of HCV in culturemedium with the patient serum at 1/50 dilution. Similar inhibition atdifferent dilutions with three other HCV infected patient sera was alsoobserved. In contrast, sera from 4 healthy individuals did not inhibitinfectivity at 1/10 dilution. These results suggested that infectiousHCV particles released in the culture medium are neutralized by specificantibodies.

REFERENCES

Applicants make no statement, inferred or direct, regarding the statusof the following references as prior art. Applicants reserve the rightto challenge the veracity of any statements made in these references,which are incorporated herein by reference.

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1) A method of producing infective hepatitis C 1a (HCV 1a) particlescomprising culturing a cell, the cell comprising, a) a human hepatocytecomprising HCV 1a cDNA, b) the HCV 1a cDNA consisting of HCV 1a cDNAencoding the HCV 1a core protein, c) the human hepatocyte beingimmortal, d) the human hepatocyte further comprising a full lengthhepatitis C virus 1a genome, e) the full length hepatitis C virus 1agenome consisting of RNA, f) the immortalized human hepatic cellreplicating the HCV 1a RNA genome and generating infective hepatitis Cvirus 1a virus particles. 2) The method according to claim 1, whereinthe full length hepatitis C virus 1a genome consisting of RNA, istranscribed from the sequence set forth in SEQ ID NO:1. 3) The methodaccording to claim 1, wherein the full length hepatitis C virus 1a viralRNA genome comprises at least 99 percent homology with RNA transcribedfrom the sequence set forth in SEQ ID NO:1. 4) The method according toclaim 1, wherein the full length hepatitis C virus 1a viral RNA genomecomprises at least 99 percent identity with RNA transcribed from thesequence set forth in SEQ ID NO:1. 5) A method of producing infectivehepatitis C 1a (HCV 1a) particles comprising, culturing a cell, the cellcomprising, a) a human hepatocyte comprising HCV 1a cDNA, b) the HCV 1acDNA consisting of HCV 1a cDNA encoding the HCV 1a core protein, c) thehuman hepatocyte being immortal, d) contacting the cell with infectiveHCV 1a particles, e) culturing the cell contacted with the infective HCV1a particles, f) the cell contacted with the infective HCV 1a particlesreplicating the HCV 1a RNA genome and generating infective hepatitis Cvirus 1a virus particles. 6) The method according to claim 5, whereinthe full length hepatitis C virus 1a genome consisting of RNA, istranscribed from the sequence set forth in SEQ ID NO:1. 7) The methodaccording to claim 5, wherein the full length hepatitis C virus 1a viralRNA genome comprises at least 99 percent homology with RNA transcribedfrom the sequence set forth in SEQ ID NO:1. 8) The method according toclaim 5, wherein the full length hepatitis C virus 1a viral RNA genomecomprises at least 99 percent identity with RNA transcribed from thesequence set forth in SEQ ID NO:1. 9) A method of screening therapeuticagents for the treatment of hepatitis comprising, a) culturing the cellof claim 1, b) contacting the cell with a with a potential therapeuticagent, c) monitoring hepatitis C virus 1a (HCV 1a) production. 10) Themethod as in claim 9, wherein monitoring HCV 1a production comprisesmeasuring replication of the HCV 1a genome using reverse transcriptaseand polymerase chain reaction. 11) The method as in claim 9, whereinmonitoring HCV 1a production comprises measuring HCV 1a polypeptideproduction using Western blots. 12) The method as in claim 9, whereinmonitoring HCV 1a production comprises the localization of virusparticles within the cell using histological methods. 13) The method asin claim 9, wherein monitoring HCV 1a production comprises thelocalization of virus particles by electron microscopy. 14) The methodas in claim 12, wherein localization of virus particles comprisesimmunochemical techniques. 15) The method as in claim 9, whereinmonitoring HCV 1a production comprises determining infectivity of HCV 1avirus particles produced by the cell.